<Emphasis Type="Italic">N</Emphasis>-acetylneuraminic acid coupled human recombinant TNFα exhibits enhanced anti-tumor activity against Meth-A fibrosarcoma and reduced toxicity

In order to study the effect of glycosylation on its biological activities and to develop tumor necrosis factor α (TNFα) with less deleterious effects, N-acetylneuraminic acid (NeuAc) with a C9 spacer was chemically coupled to human recombinant TNFα. NeuAc-coupled TNFα (NeuAc-TNFα) exhibited reduced activities in vitro by about threefold compared to native TNFα. In this study, we examined a variety of TNFα activities in vivo. NeuAc-TNFα reduced activities in the up-regulation of serum levels of IL-6 and NOx, but comparable activity as native TNFα in the down-regulation of the serum level of glucose. However, NeuAc-TNFα was more potent than TNFα in the up-regulation of the serum level of serum amyloid A (SAA). NeuAc-TNFα was less toxic to mice. In addition, NeuAc-TNFα exhibited an augmented anti-tumor activity against Meth-A fibrosarcoma without hemorrhagic necrosis. These results indicate that coupling with NeuAc enabled us to develop neoglycoTNFα with selective activities in vivo, including enhanced anti-tumor activity but reduced toxicity.     

Synthesis of glycosylated human tumor necrosis factor α coupled with <Emphasis Type="Italic">N</Emphasis>-acetylneuraminic acid

In order to study the effect of glycosylation on its biological activities, and to develop TNFα with less deleterious effects, recombinant human TNFα was chemically coupled with N-acetylneuraminic acid (NeuAc). NeuAc with C9 spacer was coupled to TNFα by acyl azide method. Two glycosylated TNFαs, designated L NeuAc-TNFα and H NeuAc-TNFα, were purified by anion-exchange chromatography. NeuAc coupling to TNFα was confirmed by lectin blotting. Average number of carbohydrate molecules introduced per molecule of L NeuAc-TNFα and H NeuAc-TNFα were estimated to be 1.0 and 1.5, respectively. We examined a variety of TNFα activities in vitro, including antiproliferative or cytotoxic activities to tumor cells, proliferative effect on fibroblast cells, stimulatory effects on IL-6 production by melanoma cells and NF-κB activation in hepatoma cells. L NeuAc-TNFα and H NeuAc-TNFα exhibited reduced activities about 1/3 and 1/10 as compared to native TNFα in all the activities performed in vitro.     

Roles of RecA protein in spontaneous mutagenesis in Escherichia coli

To verify the extent of contribution of spontaneous DNA lesions to spontaneous mutagenesis, we have developed a new genetic system to examine simultaneously both forward mutations and recombination events occurring within about 600 base pairs of a transgenic rpsL target sequence located on Escherichia coli chromosome. In a wild-type strain, the recombination events were occurring at a frequency comparable to that of point mutations within the rpsL sequence. When the cells were UV-irradiated, the recombination events were induced much more sharply than point mutations. In a recA null mutant, no recombination event was observed. These data suggest that the blockage of DNA replication, probably caused by spontaneous DNA lesions, occurs often in normally growing E. coli cells and is mainly processed by cellular functions requiring the RecA protein. However, the recA mutant strain showed elevated frequencies of single-base frameshifts and large deletions, implying a novel mutator action of this strain. A similar mutator action of the recA mutant was also observed with a plasmid-based rpsL mutation assay. Therefore, if the recombinogenic problems in DNA replication are not properly processed by the RecA function, these would be a potential source for mutagenesis leading to single-base frameshift and large deletion in E. coli. Furthermore, the single-base frameshifts induced in the recA-deficient cells appeared to be efficiently suppressed by the mutS-dependent mismatch repair system. Thus, it seems likely that the single-base frameshifts are derived from slippage errors that are not directly caused by DNA lesions but made indirectly during some kind of error-prone DNA synthesis in the recA mutant cells.     

A large-scale protein protein interaction analysis in Synechocystis sp. PCC6803.

Protein-protein interactions (PPIs) play crucial roles in protein function for a variety of biological processes. Data from large-scale PPI screening has contributed to understanding the function of a large number of predicted genes from fully sequenced genomes. Here, we report the systematic identification of protein interactions for the unicellular cyanobacterium Synechocystis sp. strain PCC6803. Using a modified high-throughput yeast two-hybrid assay, we screened 1825 genes selected primarily from (i) genes of two-component signal transducers of Synechocystis, (ii) Synechocystis genes whose homologues are conserved in the genome of Arabidopsis thaliana, and (iii) genes of unknown function on the Synechocystis chromosome. A total of 3236 independent two-hybrid interactions involving 1920 proteins (52% of the total protein coding genes) were identified and each interaction was evaluated using an interaction generality (IG) measure, as well as the general features of interacting partners. The interaction data obtained in this study should provide new insights and novel strategies for functional analyses of genes in Synechocystis, and, additionally, genes in other cyanobacteria and plant genes of cyanobacterial origin.     

Mutations in the helix termination motif of mouse type I IRS keratin genes impair the assembly of keratin intermediate filament

Two classical mouse hair coat mutations, Rex (Re) and Rex wavy coat (Re(wc)), are linked to the type I inner root sheath (IRS) keratin genes of chromosome 11. An N-ethyl-N-nitrosourea-induced mutation, M100573, also maps close to the type I IRS keratin genes. In this study, we demonstrate that Re and M100573 mice bear mutations in the type I IRS gene Krt25; Re(wc) mice bear an additional mutation in the type I IRS gene Krt27. These three mutations are located in the helix termination motif of the 2B alpha-helical rod domain of a type I IRS keratin protein. Immunohistological analysis revealed abnormal foam-like immunoreactivity with an antibody raised to type II IRS keratin K71 in the IRS of Re/+ mice. These results suggest that the helix termination motif is essential for the proper assembly of types I and II IRS keratin protein complexes and the formation of keratin intermediate filaments.     

Molecular cloning and characterization of an alpha-amylase from Pichia burtonii 15-1

An alpha-amylase secreted by Pichia burtonii 15-1 isolated from a traditional starter murcha of Nepal, named Pichia burtonii alpha-amylase (PBA), was studied. The gene was cloned and its nucleotide sequence was determined. PBA was deduced to consist of 494 amino acid residues. It shared certain degrees of amino acid sequence identity with other homologous proteins: 60% with Schwanniomyces occidentalis alpha-amylase, 58% with Saccharomycopsis sp. alpha-amylase, and 47% with Taka-amylase A from Aspergillus oryzae. A three-dimensional structural model of PBA generated using the known three-dimensional structure of Taka-amylase A as a template suggested high structural similarity between them. Kinetic analysis revealed that the K(m) values of PBA were lower than those of Taka-amylase A for the oligosaccharides. Although the k(cat) values of PBA were lower than those of Taka-amylase A for the oligosaccharide substrates, the k(cat)/K(m) values of PBA were higher.     

Haemolysin produced by Vibrio mimicus activates two Cl- secretory pathways in cultured intestinal-like Caco-2 cells

Haemolysin (VMH) is a virulent factor produced by Vibrio mimicus, a human pathogen that causes diarrhoea. As intestinal epithelial cells are the primary targets of haemolysin, we investigated its effects on ion transport in human colonic epithelial Caco-2 cells. VMH increased the cellular short circuit current (Isc), used to estimated ion fluxes, and 125I efflux of the cells. The VMH-induced increases in Isc and 125I efflux were suppressed by depleting Ca2+ from the medium or by pretreating the cells with BAPTA-AM or by Rp-adenosin 3',5'-cyclic monophosphorothioate triethylammonium salt (Rp-cAMPS). The Cl- channel inhibitors 4,4'-disothiocyanatostibene-2,2'-disulfonic acid (DIDS), glybenclamide, and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) suppressed the VMH-induced increases in Isc and 125I efflux. Moreover, VMH increased the intracellular concentrations of Ca2+ and cAMP. Thus, VMH stimulates Caco-2 cells to secrete Cl- by activating both Ca2+ -dependent and cAMP-dependent Cl- secretion mechanisms. VMH forms ion-permeable pores in the lipid bilayer that are non-selectively permeable to small ions. However, the ion permeability of these pores was not inhibited by glybenclamide and DIDS, and VMH did not change the cell membrane potential. These observations indicate that the pores formed on the cell membrane by VMH are unlikely to be involved in VMH-induced Cl- secretion. Notably, VMH stimulated fluid accumulation in the iliac loop test that was fully suppressed by a combination of DIDS and glybenclamide. Thus, Ca2+-dependent and cAMP-dependent Cl- secretion may be important therapeutic targets with regard to the diarrhoea that is induced by Vibrio mimicus.